Reagent, apparatus and method for measuring cyanuric acid

ABSTRACT

Reagents for determining presence or concentration of cyanuric acid are described. The reagents include an indicator, a 2,4-diamino-6-alkyl-1,3,5-triazine, and may include a stabilizer, an antioxidant, or both.

FIELD OF THE INVENTION

This invention relates to apparatus, methods, and reagents useful inanalyzing liquid samples. More particularly, the invention relates toanalyzing liquid samples to determine cyanuric acid in such a liquidsample.

BACKGROUND AND PRIOR ART

The science of analytical chemistry has, and continues to make progress.The field involves the ability to assay sample materials to determine ifa particular substance or substances are present, and if so, the amountof that substance. Frequently, the term “analyte” is used to describethe substance being tested. This term will be used hereafter.

Early examples of the application of analytical chemistry include litmuspaper, as well as devices which would change color if atmospherichumidity was above a particular level. To say that the field has becomemore sophisticated since then is an understatement.

One area of importance in analytical chemistry is the testing andevaluation of liquid samples. While “liquid sample” as used hereafterrefers to materials such as blood, urine, but most particularly for thisdisclosure, water.

It is desirable and necessary to analyze water for various components.For example, it may be important to determine if a water sample ispotable. Further, water samples are used for different purposes.Depending upon the use to which the sample is to be put, one or moreparameters, such as pH, total alkalinity, calcium hardness, totalhardness, and amount of particular analytes such as total chlorine, freechlorine, combined chlorine, sodium content, etc., may be important. Forexample, when the water sample is taken from a swimming pool, either orboth of combined chlorine and free chlorine may be important. Where thewater is to be used for an industrial cooling system, total alkalinityor total hardness may be important. When the water is to be used in thehealth profession, any number of analytes may be of interest andimportant. These are just examples of the type of uses to which watersamples may be put. The skilled artisan will be familiar with manyothers, which need not be set forth here. Further, the literature onanalysis of liquid samples other than water is vast.

Analysis of water samples can be accomplished with any number ofdifferent systems. Generally, however, these systems can be divided into“dry chemistry” and “wet chemistry” systems.

In a wet chemistry system, essentially one adds either a liquid testingagent or a dissolvable testing agent to a liquid sample. The testingagent reacts with the analyte of interest, leading to formation of adetectable signal. Preferably, this is the formation of a visible“marker,” such as a color or change in color. Again, the artisan will befamiliar with other systems such as measurement of light absorption inphotometers, etc. For purposes of this disclosure, however, thediscussion will focus on visible formation and changes in color, ratherthan systems such as light photometers solely to facilitateunderstanding.

In these wet chemistry systems, the reacted liquid sample is thencompared to some reference standard. Generally, this takes the form of acoded reference linking concentration of the analyte to a particularcolor or degree of color. A low concentration may be indicated by a verypale pink color, and a high concentration by one which is dark red, andvice versa.

Dry chemistry systems can be used to analyze many of the types ofsamples that wet chemistry systems are used to analyze. In these drychemistry systems an apparatus, such as an absorbent pad or a test stripis impregnated, coated, or printed with the test system discussed supra,in such a way that the test system does not and cannot leave theapparatus. The apparatus is contacted with the liquid sample, removedfrom it, and the signal is “read” on the apparatus. As with wetchemistry systems, the signal that is generated is compared to a codedreference to link the signal generated to a specific amount and/orconcentration of an analyte under consideration.

The prior art literature on analytical chemistry is vast. For example,U.S. Pat. No. 5,811,254, to Wu, teaches reagent systems which can beused to detect total available chlorine over an extensive range (0 to5000 ppm). The reagents can be incorporated into a carrier matrix, suchas filter paper, to produce a dry chemistry test strip useful inmeasuring total available chlorine. U.S. Pat. No. 5,710,372, to Becket,teaches test strips which include a plurality of test regions. Eachregion contains a different amount of a reagent system which reacts withan analyte of interest. A visual display results which permits the userto determine the amount of the analyte in the sample being analyzed.U.S. Pat. No. 5,620,658, to Jaunakais, teaches multicomponent teststrips which contain reagents capable of converting undetectableanalytes into detectable ones, via ionic change. U.S. Pat. No.5,529,751, to Gargas, teaches a pH adjustment kit. Once the pH of thesample has been determined, a first reagent is added until the sampleindicates that a proper pH has been obtained. The number of drops of thefirst reagent is then converted to a quantity of a second reagent, whichis then used to modify pH of the source of the sample. U.S. Pat. No.5,491,094, to Ramana, et al., teaches dry reagent test strips fordetermining free chlorine, using TMB derivatives. U.S. Pat. No.4,904,605, to O'Brien, et al., teaches test strips which can be used todetermine a plurality of different reagents. A dipstick containing aplurality of reagent pads is contacted to sample, signal is formed, andthen compared to a reference standard. U.S. Pat. No. 4,481,296, toHalley, teaches compositions that are useful in determining the pH of ahalogen containing solution.

As the number of swimming pools and spas increases, the need foreffective tools to monitor and control pool water chemistry andespecially sanitizer levels becomes more and more important. This isespecially true in pools used by the public where the batherconcentration is high and the threat of contagious diseases is alwayspresent. In order to control the harmful microorganism population ofpools, it has been found over the years that chlorine is the mosteffective and economical sanitizer. However, as popular as chlorine is,it nevertheless has certain drawbacks which must be considered. Aparticularly serious problem associated with the use of chlorine inoutdoor pools is that it tends to be destroyed by sunlight.

In this regard it has been found that the addition of cyanuric acid(2,4,6-trihydroxy-1,3,5-triazine) to pool water can be effective as anextender or stabilizer for chlorine. However, the concentration must berather carefully adjusted since too little obviously is ineffective as astabilizer for the chlorine while too much can dramatically slow downthe rate at which microorganisms are destroyed by the chlorine. It hasbeen found that the effective concentration of cyanuric acid liesbetween 30 and 100 parts per million (ppm).

In order to maintain the effectiveness of the cyanuric acid in theswimming pool, it is necessary to measure the concentration thereofusing a test device or concentration measuring system. The current testmost commonly used in the swimming pool industry involves the melamineturbidimetric method. In this scheme, melamine is added to a sample ofthe pool water which, in the presence of cyanuric acid, causes theformation of a finely dispersed precipitate. The turbidity created bythis precipitate formation is proportional to the amount of cyanuricacid present. By measuring this turbidity using visual or instrumentalschemes, an estimation of the concentration of cyanuric acid can beobtained. This test however is not completely acceptable sinceturbidimetric methods tend, in general, to be unreliable in that otherfactors can cause turbidity and precipitates are obviously lesshomogenous than solutions.

For this reason, attempts have been made over the years to replace theturbidimetric analytical procedures with colorimetric methodologies.

Various approaches have been suggested for determining cyanuric acid insamples. For example, U.S. Pat. No. 2,986,452 to Merek suggests theaddition of sodium acetate and a soluble copper salt. Mancini, U.S. Pat.No. 4,039,284, utilized a combination of thymolsulfonphthalein andmonoethanolamine.

With Stillman, U.S. Pat. No. 4,793,935, more modern systems can be seen.This patent teaches that cyanuric acid, when reacted with melamine,forms a precipitate thus removing the cyanuric acid from water. It isnot an analytical method.

U.S. Pat. No. 4,855,239 to Rupe uses melamine as a component of anindicator system for determining cyanuric acid, and Fernando, U.S. Pat.No. 6,432,717, involves an improvement on this earlier patent using astabilizing compound. Ghanekar, published Application U.S. 2003/0147777,uses an indicator which changes color in response to a change in pH as away to determine cyanuric acid.

Melamine is somewhat structurally related to the compoundacetoguanamine, or 2,4-diamino-6-methyl-1,3,5-triazine, differing inthat melamine has an amino group at position 6, whereas acetoguanaminehas a methyl group.

While there is this element of structural similarity, it has been foundthat, among compounds structurally related to melamine, the 6-alkylderivatives, acetoguanamine, in particular, are useful in cyanuric acidassays, as will be shown in the disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

The feasibility of using 2,4-diamino-6-methyl-1,3,5-triazine (“DAMT”hereafter) was tested in solution based assays.

A stock solution of cyanuric acid (2000 ppm) was prepared and thendiluted to 40, 100, 200, and 300 ppm. A reagent blank with 0 ppmcyanuric acid (“CYA” hereafter) was also used. The pH of each solutionwas adjusted with 0.1N HCl to 6.8 and the indicator phenol red wasadded. DAMT was then added, and any color changes were monitoredvisually.

Gradations from yellow (0 ppm CYA) to reddish purple (300 ppm CYA) wereobserved, suggesting that DAMT could be used to differentiate CYAlevels.

EXAMPLE 2

The results, supra, suggested testing phenol red, as well as theindicator cresol red in dry paper test strips.

Formulations of DAMT in deionized water (1%) were prepared.

Indicator dips were prepared by mixing 8.0 ml of a 0.5% solution of thesodium salt of one of the indicators mentioned supra, 67.5 ml (0.675%)of the DAMT solution, and 24.5 ml of deionized water. The solution ofphenol red was adjusted to pH 6.8, and that of cresol red, to pH 7.2.

Each solution was then used to impregnate a variety of filter papers,which were then dried, at 90° C., and tested in CYA solutions, preparedfrom the stock standard CYA solution, as discussed supra, with theexception that the pH of the standard solution was adjusted to 7.5, andthe pH of test solutions was not adjusted.

Several of the test strips gave good results, with clear colordemarcations at the different CYA concentrations.

In an attempt to improve the product, the experiments described suprawere repeated, with the pH adjusted up to 7.5 for cresol red, and 7.2for phenol red.

All tests gave good results.

EXAMPLE 3

The impact the pH of a solution had on the test was studied, byadjusting the pH of the 0 ppm CYA solution to about 7, and about 8. Thedipsticks, as described supra, were tested, and compared to the firstset of results.

Phenol red strips, when dipped in the 0 ppm standard at 7.0, gave afalse positive reading of about 70 ppm CYA, and about 100 ppm whendipped in the 0 ppm standard, at pH 8. Cresol red strips gave resultscomparable to about 60 and 80 ppm, respectively.

These results suggested that it would be desirable to modify the stripsslightly to avoid false positives.

EXAMPLE 4

A reagent solution was prepared by combining 5 ml 0.4% cresol red sodiumsalt, 50 ml 1.3% DAMT, and adjusting it to a pH 7.4 with 0.1N HCl. Astrip of paper (1.5″×6″) was immersed in the solution and excess liquidwas removed with a stirring rod. The end was touched off on an absorbenttowel. The paper was dried for 30 minutes at 60° C. The dried paper wasattached to 3M double stick tape, one/fifth inch of the edges wasdiscarded, and cut into ⅕ inch strips. The backing was peeled off andthe paper with double stick tape was mounted on a plastic card near thelong edge, and the card was cut into ⅕ inch test strips.

Test solutions were prepared by adjusting tap water with sodiumbicarbonate to give an alkalinity of 100 ppm, then a 2000 ppm cyanuricacid solution was diluted with the 100 ppm water to give cyanuric acidsolutions of 0, 50, 100, 200, and 300 ppm cyanuric acid. The pH of eachsolution was adjusted to 7.4 with 0.1N HCl.

The cyanuric acid solutions were then tested with the above test strips.The test strips gave easily distinguishable colors ranging fromtan-yellow to red purple. All levels were easily distinguishable fromadjacent levels.

EXAMPLE 5

Test strips were prepared as in example 1, except2,4-diamino-6-methyl-1,3,5-triazine was replaced by 0.6%2,4-diamino-6-n-butyl-s-triazine. The test strips were tested as inExample 4. The range of color was not as large as in example one, butadjacent levels could be distinguished.

EXAMPLE 6

The example describes the manufacture of cyanuric acid determiningdipsticks, as optimized.

A formulation (1 liter) was mixed which contained:

Cresol red, sodium salt 0.5 g DAMT 12.0 g Sodium thiosulfate, 5 H₂O 0.5g Propylene glycol 40 ml Deionized water 960 ml

The pH of the solution was 6.9. The sodium thiosulfate is an antioxidantwhich inhibits halogenation from, e.g., chlorine when a halide orhalogen is present in the test sample. The propylene glycol acts tostabilize the mixture and to facilitate even application to the paper.Using this formula, the test paper was impregnated, and dried.

Strips thus prepared were tested with CYA standards adjusted to 100 ppmtotal alkalinity, as described supra, and showed color differentiationat different concentrations.

EXAMPLE 7

The compound ethylenebismelamine, described in U.S. Pat. No. 5,514,213,incorporated by reference, was tested as described, supra, and alsoshowed differentiation at varying concentrations.

The foregoing examples describe the features of the invention, whichrelate to the use of 2,4-diamino-6-alkyl-1,3,5-triazines in thepreparation of reagents and apparatus useful in determiningconcentrations of cyanuric acid in fluids, such as liquids, includingwater, such as swimming pool water, sauna or spa water, and so forth.The alkylene moiety in these compounds may be straight or branched,substituted or unsubstituted, and contain from 1 to 10 carbon atoms,more preferably 1 to 6 carbon atoms, and is most preferably straightchained, unsubstituted, containing from 1 to 4 carbon atoms.

The compound described supra is combined with an indicator molecule, andmay optionally contain a substance, which inhibits interference by otheranalytes, such as an antioxidant which inhibits halogenation, and/or astabilizer. Exemplary of indicators are cresol red, phenol red, thymolblue, m-cresol purple, or any of the other indicators well known in theart, which exhibit a visible color change or formation of color at thepH range. Further examples include nitrazine yellow, bromothymol blue,bromophenol blue, brilliant yellow, neutral red, 3-nitrophenol, orangeII, phenolbenzein (aurin), cresolbenzien, and other indicators wellknown to the artisan. The interference inhibitor may be an antioxidantsuch as sodium thiosulfate, sodium metabisulfite, sodium bisulfite,sodium sulfite, ascorbic acid, 3-tert-butyl-4-hydroxyanisole,2,6-di-tert-butyl-4-methylphenol or other antioxidants well known in theart. The stabilizer is preferably propylene glycol, but the skilledartisan will be aware of other useful stabilizers, such as ethyleneglycol, other alkylene glycols, and so forth.

The reagent is formulated so as to have a pH of from about 6.0 to about9.0, more preferably from about 6.5 to about 8.0, and most preferably,from about 6.5 to about 7.5. The reagents may be in liquid form, or as acombination of dry ingredients, or may be formulated as kits, wherein acontainer means affords a holding device for the components, which arekept separate from each other until used by the artisan.

The reagents may be applied to a solid substrate, such as a bibulouspaper, or other absorbent or absorbent medium, and then dried, so thatthey can be used, e.g., as dipsticks to analyze cyanuric acid content ofsamples. One may use oxygenated solvents, like MEK or MIBK to increasesolubility of the triazine and permit deposition of more reagentthereon.

Solid substrates containing the reagent of the invention may beformulated so that the apparatus contains only the reagent describedsupra, or may be a component of a solid substrate which measures otherparameters as well.

The terms and expression which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. Reagent useful in determining cyanuric acid in a fluid sample,comprising a 2,4-diamino-6-alkyl-1,3,5-triazine or ethylenebismelamine,wherein said alkyl contains from 1 to 10 carbons, is straight chained orbranched, substituted or unsubstituted, and an indicator molecule, at apH of from about 6 to about
 9. 2. The reagent of claim 1, wherein saidreagent is at a pH of from about 6.5 to about
 9. 3. The reagent of claim1, further comprising a stabilizer.
 4. The reagent of claim 3, whereinsaid stabilizer is propylene glycol.
 5. The reagent of claim 1, furthercomprising an antioxidant.
 6. The reagent of claim 5, wherein saidantioxidant is sodium thiosulfate.
 7. The reagent of claim 1, furthercomprising an antioxidant and a stabilizer.
 8. The reagent of claim 7,comprising cresol red, 2,4-diamino-6-methyl-1,3,5-triazine, propyleneglycol, and sodium thiosufate.
 9. The reagent of claim 1, wherein saidalkyl is straight chained and consists of 1 to 4 carbon atoms.
 10. Thereagent of claim 1, wherein said reagent comprises2,4-diamino-6-methyl-1,3,5-triazine.
 11. The reagent of claim 1, whereinsaid indicator is cresol red or phenol red.
 12. Apparatus comprising thereagent of claim 1, impregnated, absorbed or absorbed onto a solidcarrier.
 13. The apparatus of claim 12, wherein said solid carrier isabsorbent or absorbent paper.
 14. The apparatus of claim 13, furthercomprising at least one other reagent suitable for determining a secondanalyte wherein said second analyte is present in said fluid samplebeing tested for presence of cyanuric acid.
 15. A method for determiningcyanuric acid in a fluid sample, comprising contacting said sample withthe reagent of claim 1 and determining formation of or change of a coloras an indication of presence or concentration of cyanuric acid in saidfluid sample.
 16. The method of claim 15, wherein said fluid sample isswimming pool water.